摘要
目的构建日本血吸虫动力蛋白轻链DNA疫苗和小鼠干扰素真核表达质粒,研究其在HeLa细胞及动物体内的表达。方法设计特异性引物,PCR扩增获取动力蛋白轻链(SjDLC)与干扰素-γ(IFN-γ)基因,克隆于pcDNA3.1真核质粒,经双酶切及测序鉴定。将两种重组质粒分别转染至HeLa细胞,Western blot鉴定其表达。真核质粒经左腿股四头肌免疫小鼠,在末次免疫2周后,PCR法检测小鼠肌组织内SjDLC和IFN-γ基因,免疫组织化学法检测基因的表达,MTT法检测T细胞增殖水平。结果 PCR和双酶切能检测到280 bp的SjDLC基因及480 bp的IFN-γ基因;Western blot显示在12 kDa和19 kDa处有阳性反应条带,与SjDLC和IFN-γ分子量大小一致,两种基因能在HeLa细胞中表达。PCR及免疫组化显示两种基因能在小鼠肌肉组织中存在和表达。MTT法显示两种质粒均能刺激T细胞增殖。结论成功构建pcDNA3.1/SjDLC和pcDNA 3.1/mIFN-γ真核质粒,两种质粒能在HeLa细胞和动物体内表达。
Objective To construct the dynein light chain of Schistosoma japonicum DNA vaccine and IFN-γ recombi- nant plasmid of mice,so as to investigate their expression in HeLa cells and mouse. Methods A pair of primers designed by PRIMER5.0 software was synthesized;the gene fragments of SjDLC and mIFN-γ/were amplified by PCR, and inserted into eukaryotic expression plasmid pcDNA3.1, where the recombinant plasmids were identified with restrictive enzymes and sequenced.The recombinant vectors pcDNA3.1/SjDLC and pcDNA3.1/mIFN-γ/were transfected into HeLa cells;the expressed proteins were identified by Western blot.These plasmids were injected into BALB/c mice too ,the gene of SjDLC and IFN-γ/in quadriceps femoris were identified by PCR. The expression of SjDLC, IFN-γ were observed with immunohistochemistry. The proliferation of splenic lymphocytes in mice was detected by MTT. Results The 280 bp of SjDLC gene and 480 bp of IFN-γ gene were observed through PCR and double enzyme digestion ;Western blot displayed that positive response bands in 12 kDa and 19 kDa which is consistent with the molecular weight of SjDLC and IFN-γ were successfully expressed in HeLa cells.PCR and immunohistochemistry showed that two genes expressed in mouse muscle tissue.MTT showed that two plasmids could stimulate the proliferation of T cells. Conclusion The peDNA/SjDLC DNA vaccine and recombinant plasmid pcDNA/mIFN-γ/were successfully constructed and then expressed in HeLa cells and mouse.
出处
《中南医学科学杂志》
CAS
2017年第6期558-561,579,共5页
Medical Science Journal of Central South China
基金
湖南省教育厅科学研究项目(编号:13C824)
湖南省研究生科研创新项目(CX2015B415)
湖南省分子靶标新药研究协同创新中心资助项目(NO.2015-351)
特殊病原体防控湖南省重点实验室资助项目(NO.2014-5)
